Detection and treatment of pregnancy complications comprising determining sialyl Lewis antigens and administering hCG

ABSTRACT

Disclosed herein is a method of identifying and/or addressing incipient preeclampsia in a patient-subject by the steps of (a) performing a bioassay to determine the level of at least one sialyl Lewis antigen in a said patient-subject at about 25 weeks of pregnancy or earlier; (b) performing a bioassay to determine the level of at least one sialyl Lewis antigen in a pregnant non-preeclampsia one or more subjects at about 30 weeks of pregnancy or later, wherein said at least one sialyl Lewis antigen assay is for a sialyl Lewis antigen assayed in step (a) is and if more than one subject is assayed, averaging said results; and (c) managing said patient-subject for preeclampsia, if said level of at least one sialyl Lewis antigen of step (a) is at or greater than about 20% above the level of such sialyl Lewis antigen assayed in step (b).

RELATED APPLICATION DATA

This application is a continuation of U.S. application Ser. No.15/889,261, filed Feb. 6, 2018, which is a continuation of U.S.application Ser. No. 14/438,033, filed Apr. 23, 2015, which is the U.S.national phase pursuant to 35 U S C. § 371, of International applicationSer. No. PCT/US2013/067032, filed Oct. 28, 2013, which claims thebenefit of U.S. provisional application Ser. No. 61/721,228, filed Nov.1, 2012, and 61/739,817, filed Dec. 20, 2012. The entire disclosures ofthe aforementioned patent applications are incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates to a method of identifying and/oraddressing incipient preeclampsia in a patient-subject by the steps of(a) performing a bioassay to determine the level of at least one sialylLewis antigen in a said patient-subject at about 25 weeks of pregnancyor earlier, (b) performing a bioassay to determine the level of at leastone sialyl Lewis antigen in one or more pregnant non-preeclampsiasubjects at about 30 weeks of pregnancy or later, wherein said at leastone sialyl Lewis antigen assay is for a sialyl Lewis antigen assayed instep (a) is and if more than one subject is assayed, averaging saidresults; and (c) managing said patient-subject for preeclampsia, if saidlevel of at least one sialyl Lewis antigen of step (a) is at or greaterthan about 20% above the level of such sialyl Lewis antigen assayed instep (b).

Particular reference is made to sialyl Lewis type antigens onglycoproteins.

Also noted are assays performed in liquid samples. A particularimmunoassay is used as diagnostic tool to predict onset of pregnancycomplications including preeclampsia and HELLP syndrome (i.e., a groupof symptoms that occur in pregnant women characterized by hemolysis,elevated liver enzymes, and low platelet count). A further aspect is useof glycoprotein devoid of blood group sialyl Lewis type antigens torescue, prevent or treat preeclampsia-like symptoms.

BACKGROUND

The following abbreviations are used herein:

BMDC—bone marrow derived dendritic cells

hCG—human chorionic gonadotropin

HhCG—hyperglycosylated hCG

IDO—indoleamine 2,3-dioxygenase

IFN-γ—interferon gamma

Tregs˜regulatory T cells

Th17—T helper 17 cells

TGF—transforming growth factor

TF—Thomsen-Friedenreich antigen

uNK—natural killer cells

IL-10 Interleukin 10 VEGF C—Vascular endothelial growth factor C

VEGF C—Vascular endothelial growth factor C

NPS—normal pregnancy serum

PE preeclampsia

PES preeclampsia serum

sEng—soluble endoglin

GPs—gestational pathologies.

Pregnancy is a dynamic process characterized by immune tolerance,angiogenesis and hormonal regulation. Human chorionic gonadotropin (hCG)is reported as detected on the first day of implantation; its levelspeak around gestational week 12 and diminish to low levels during theremainder of pregnancy. hCG is believed to exhibit a number of functionsin pregnancy, including the promotion of progesterone production,implantation and decidualization, angiogenesis, cytotrophoblastdifferentiation, and immune cell regulation. With these myriad functionsin mind, hCG dysregulation could lead to adverse pregnancy outcomes.Preeclampsia is a condition marked by insufficient trophoblast invasionand maternal spiral artery remodeling and inflammation. Recent studieshave reported a link between preeclampsia and immune cell dysregulation,including reduced numbers of uterine and circulating regulatory T cells(Tregs) and natural killer (uNK) cells.

Managing women for preeclampsia is a topic widely addressed in the art.By way of non-limiting example, reference is made to The Diagnosis andManagement of Pre-eclampsia and Eclampsia, O'Loughlin et al. (Instituteof Obstetricians and Gynaecologists, Royal College of Physicians ofIreland (Version 1.0. Guideline No. 3, 2011).

hCG Variants

hCG is composed of a and β subunits each consisting of a proteinbackbone with N-linked and O-linked oligosaccharides. It is now believedthat there are four distinct variants: hCG, hyperglycosylated hCG(HhCG), the free β-subunit, and pituitary hCG. These four can be furthermodified by partial degradation of the hCG molecule, nicking of theintact β-subunit, or variation of the attached

Oligosaccharides

These variants play different roles in both normal and abnormalpregnancy. HhCG, has complex β-subunit N- and O-linked oligosaccharidesstructural alterations. HhCG is produced early normally duringpregnancy; it does not have high affinity to LH/hCG receptors, yet isreported to promote invasion and growth of cytotrophoblasts byinteracting with transforming growth factor (TGF) β receptors. After theinitial three to four weeks of pregnancy, the levels of HhCG become verylow and hCG (non-hyperglycosylated) is usually the predominant formRecent studies have reported additional variants with distinctsialylated oligosaccharides of the Lewis type pattern on hCG isolatedfrom serum of pregnant women. Differential expression of suchcarbohydrates is associated with inhibition of E-selectin-mediatedhoming of leukocytes and may contribute to early pregnancy loss throughpoor placental-immune interactions. Without being bound by anyparticular theory, it is believed that in the time between placentationand parturition a dynamic structural conversion of one form of hCG toalternate forms of hCG is choreographed. This suggests that impairmentor alterations in hCG glycosylation patterns affect its signaling andbiological activities.

hCG, Angiogenesis and Immune Tolerance

The maternal-fetal interface is replete with immune cells whichcross-talk with hormonal, endocrine, and angiogenic regulators toprogram a normal pregnancy outcome. Among immune cell types, regulatoryT cells a specialized CD4 T cell subset phenotyped as CD4+CD25+/Foxp3+,play an important role in protecting the fetus by dampening harmfulinflammatory immune responses at the maternal-fetal interface. It hasbeen shown in humans that Treg numbers increase very early in pregnancy,peak during the early second trimester and then begin to decline untilthey reach pre-pregnancy levels. Tregs have also been described assignificant in immune tolerance of the fetus in the mouse pregnancymodel. Tregs have been described as following a gestationalage-dependent presence in the uterus. Animal studies further indicatethat tolerance to paternal antigens may be initiated during mating whenseminal fluid and components of semen have been shown to triggerexpansion of the Treg cell population. Further, it has been reportedthat Tregs migrate toward areas of hCG production, indicating that innormal pregnancy, these cells may be attracted to hCG produced bytrophoblasts at the maternal-fetal interface ensuring immune toleranceof the fetus. However, if hCG undergoes dysregulation during pregnancy,its control over immune tolerance pathways may be impaired.Interleukin-10 (IL-10) and the tryptophan-metabolizing enzymeindoleamine 2,3-dioxygenase (IDO) are two specific immune regulatorsLevels of IL-10, an immunosuppressant, reportedly increase in earlypregnancy and remain elevated until the onset of labor, possiblyregulating maternal immunity and allowing acceptance of the fetalallograft. IL-10 reportedly regulates uNK cell maintenance and furthercontrols their cytotoxic functions in response to pro-inflammatorychallenges during pregnancy. Further, decidual Tregs reportedly inhibitimmune stimulation of T cells through IL-10 production. It is believedthat the temporal expression of IDO regulates the Tregs and preventsthem from being converted to pro-inflammatory Th17 cells hCG reportedlystimulates IL-10 production of murine BMDC. This same study found thattreatment of BMDC with hCG and interferon gamma (IFN-γ) increased IDOmRNA production and enzyme activity.

It is noteworthy that hCG is now considered as an angiogenic factor andthus may regulate an endovascular cross-talk between trophoblasts,endothelial cells, and immune cells represented by uNK cells. In animalstudies these specialized cells have been reported as playing a role inspiral artery remodeling and trophoblast invasion Vascular endothelialgrowth factor C (VEGF C) production by uNK cells is responsible fortheir non-cytotoxic activity, and that VEGF C producing uNK cellssupport endovascular processes in vitro. It is possible that thetolerogenic phenotype of uterine NK cells during early decidualizationis be influenced by hCG through stimulation of the quiescent angiogenicmachinery. Recent studies indicate that the uNK cells are indeedinfluenced by hCG. One study reported that hCG induces proliferation ofhuman uNK cells, by interacting through the mannose receptor rather thanthe LH/hCG receptor.

hCG and Preeclampsia

Some reports define preeclampsia as hypertension in a previouslynormotensive pregnant female and proteinuria after 20 weeks of pregnancy(or gestation). Preeclampsia affects 5-10% of all pregnancies andremains a leading cause of maternal and fetal morbidity and mortality.Related gestational pathology include Intrauterine growth restriction,eclampsia, gestational hypertension (i.e., hypertension in pregnancywithout proteinuria) broadly termed “gestational pathologies” (“GPs”).

Based on clinical presentation, preeclampsia is considered as a latepregnancy disorder. Molecular events leading to its onset seem to occurearlier in pregnancy Portrayed as a two stage disorder, maternalsymptoms of preeclampsia are classified as consequences of pre-clinicalplacental pathology associated with poor placental perfusion,inflammation, ischemia/hypoxia, and trophoblast damage. Despite thepro-angiogenic role of hCG, little is known about the endovascularinteractions of trophoblasts and endothelial cells and its subsequenteffects on spiral arteries especially in the presence of different formsof hCG. Recently we reported that injection of preeclampsia serum inpregnant IL-10⁷″ mice results in hypertension and proteinuria. Thetreatment also led to a perturbed immune cell population at thematernal-fetal interface. Higher hCG levels in preeclampsia serum atterm as compared to normal pregnancy serum has been reported. Severalstudies have reported a decrease in Treg cell population both in thecirculation and in placental bed sections in preeclamptic women ascompared to those with normal pregnancy. Since IL-10 and hCG areimplicated in normal pregnancy outcome, it is tempting to speculate thatdeficiency in these molecules may predispose to severe preeclampsiapathology. Animal studies from our lab suggest that IL-10 deficient miceare more sensitive to serum- and hypoxia-induced onset ofpreeclampsia-like features.

All documents cited herein are incorporated by reference in theirentirety as if fully set forth herein.

Reference is made to the following:

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SUMMARY OF THE INVENTION

Disclosed herein is a method of determining incipient preeclampsia at anearly stage of pregnancy. Early is understood to include from about 20weeks of pregnancy or earlier, or about 15, weeks or earlier or 10 weeksor earlier. The method comprises the steps of

(a) performing a bioassay to determine the level of at least one sialylLewis antigen in a said patient-subject at about 25 weeks of pregnancyor earlier;

(b) performing a bioassay to determine the level of at least one sialylLewis antigen in a pregnant non-preeclampsia one or more subjects atabout 30 weeks of pregnancy or later, wherein said at least one sialylLewis antigen assay is for a sialyl Lewis antigen assayed in step (a) isand if more than one subject is assayed, averaging said results;

(c) managing said patient-subject for preeclampsia, if said level of atleast one sialyl Lewis antigen of step (a) is at or greater than about20% above the level of such silalyl Lewis antigen assayed in step (b).

In some embodiments of this method the above noted determination ofincipient preeclampsia is made at about at 25 weeks of pregnancy orearlier; about 15 weeks or earlier, 12 to 14 weeks or earlier, at about10 weeks or earlier and at about 1 week. In some embodiments thedetermination of step (b) with diagnoses of either pre-eclampsia ornon-preeclampsia female is made at about 32 weeks of pregnancy or later.

Also disclosed is a method of determining comparators of a diagnosis ofincipient preeclampsia at 20 weeks of pregnancy or earlier by the stepsof

(a) determining the level of sialyl Lewis antigen in a female subjectwith a diagnosis of preeclampsia about 30 weeks of pregnancy or later,and,

(b) determining the level of sialyl Lewis antigen in a pregnantnon-preeclampsia female subject at about 30 weeks of pregnancy or later.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of serum and amniotic fluid hCG exhibiting expressionof sLex and sLea as compared to urine hCG and hCG secreted insupernatants from choriocarcinoma cells BeWo or Jeg 3.

FIG. 2. is a graph of sialyl Lewis antigen (sLe^(Y), sLe^(a) andsLe^(x)) expression on preeclampsia serum (PES)-hCG as compared tonormal pregnancy serum (NPS)-hCG.

FIG. 3 is a graphic quantification of sialyl Lewis antigens in serum.

FIG. 4A are displays fetal size (upper panel) and fetal weights (lowerpanel).

FIG. 4B presents blood pressure data

FIG. 4C presents mouse subject proteinuria levels.

FIG. 5A shows renal pathology by H&E staining of the glomerulus

FIG. 513 is a graph of production of sFlt-1.

FIG. 5C is a graph of sEng in pregnant IL-10⁷″ mice.

DETAILED DESCRIPTION OF THE INVENTION

In the practice of this invention it is to be understood that thedetection of PE (or related GPs) in instances where the pathology iseffectively treated is the detection of precursor indicia before 20weeks of gestation. For convenience the notation “sLe<20w” is used todenote sialyl Lewis antigen elevation before 20 weeks of pregnancy. Inthis context, “elevation” will be understood to mean up-regulation orincrease of at least about 20% of each of sialyl Lewis antigen alone ascompared to the average control of the respective sialyl Lewis antigen,or more than 30% if two sialyl Lewis antigen levels are combined, and50% increase if three are combined. There are a number of bioassayssuitable to determine such levels and other assays are being developed.

The practice of the invention, in one embodiment, is characterized byfinding altered carbohydrate patterns, with specific reference to by theexcess or elevated presence of sialyl Lewis antigens on preeclampsia hCGas compared to normal pregnancy hCG Another aspect is the therapeuticuse of sialyl Lewis antigen-free hCG in mitigating the symptomsassociated with pregnancy complications such as preeclampsia. (ExpertOpin Drug Deliv. 2012 August; 9(8):893-900. Epub 2012 Jun. 18.)

hCG Can Rescue Pregnancy.

Without being bound by any particular theory, in IL-10″ mice, it isbelieved that the mode of action is by subverting production ofanti-angiogenic factors and by replenishing uterine immune cells.Deglycosylated hCG is not reported as able to bind to mannose receptorson uNK cells, again emphasizing the importance of carbohydrate patternsin the function of hCG. Given the functional associations co-regulatedby hCG, IL-10 and Treg migration, dysregulated hCG effects uterine Tregsand contributes to preeclampsia. Particularly noted is therapeuticadministration of CG. CH with a less antigenic presentation is useful.This includes recombinant hCG. Intravenous administration is noted.Dosing with recombinant hCG, i.v. from between about 50 I.U. to about500 I.U with particular reference to dosages between about 100 I.U and200 I.U. is noted. Prefilled pens for administration of recombinanthuman chorionic gonadotropin (r-hCG) are available and useful in thepractice of this invention.

EXEMPLIFICATION Example 1 Quantification of Sialyl Lewis Antigens on hCGin Different Biological Fluids

Ninety-six-well microtitre plates (Maxisorp, Nunc) were coated with 50μI rabbit anti-human hCG antibody (5 μg/ml in PBS, Dako A0231) at 4° C.overnight. The wells were washed three times with PBS, pH 7.2 containing0.05% Tween 20, blocked for 1 hour with washing buffer containing 1% BSAand washed again three times.

50 μl of pregnancy serum/amniotic fluid/urine/cell culture supernatantsamples or hCG (5 μg/ml) were added and incubated for 1.5 hours at roomtemperature and washed three times. 50 μl of sLe^(x) (Calbiochem, KM93)or sLe^(a) (Calbiochem, KM 231) recognizing antibodies were added atconcentration of 1 μg/ml. The wells were incubated for 1.5 hours at roomtemperature and washed three times 50 μl of HRP-conjugated rabbitanti-mouse antibody (Dako, PO260) was added to each well, incubated for1.5 hours, washed three times, developed with DMB and color developmentwas followed by measuring the absorbance at 492 nm/630 nm Wells withouthCG served as controls.

Example 2 Quantification of Sialyl Lewis Antigens from PreeclampsiaSerum hCG and Normal Pregnancy Serum hCG

Ninety-six-well microtitre plates (Maxisorp, Nunc) were coated with 50μI rabbit anti-human hCG antibody (5 μ9/ιτιI in PBS, Dako A0231) at 4°C. overnight. The wells were washed three times with PBS, pH 7.2containing 0.05% Tween 20, blocked for 1 hour with washing buffercontaining 1% BSA and washed again three times.

50 μI of human normal (n=15) or preeclampsia diagnosed pregnancy serum(n=14) obtained from blood collected at 32-36 weeks of pregnancy wereadded and incubated for 1.5 hours at room temperature and washed threetimes.

50 μI of sLe^(x) (Calbiochem, KM93) or sLe^(a) (Calbiochem, KM 231) orLe^(y) or Thomsen-Friedenreich antigen (Glycotope) recognizingantibodies were added at concentration of 5 μg/ml in PBS. The wells wereincubated for 1.5 hours at room temperature and washed three times.

50 μI of HRP-conjugated rabbit anti-mouse antibody (Dako, PO260) wasadded to each well, incubated for 1.5 hours, washed three times,developed with DMB and color development was followed by measuring theabsorbance at 492 nm/630 nm. The mean absorbance obtained with multiplenormal pregnancy hCG were considered as 100%.

As seen in the FIG. 2, sialyl Lewis antigen (LeY, sLeA and sLeX)expression is significantly higher on preeclampsia serum (PES)-hCG ascompared to normal pregnancy serum (NPS)-hCG As seen in FIG. 2, therewas 21.6% up-regulation of sLe^(a) (P=0.002), 32% up-regulation ofsLe^(x) (P=0.019) and 21.6% up-regulation of Le^(Y) in the 32nd-36thweek of gestation (P=0.021) in preeclamptic hCG compared to normalpregnancy hCG. The increase in sialyl Lewis antigen expression inPES-hCG was independent of the serum levels of β-hCG.

Example 3 Quantification of Sialyl Lewis Antigens in Serum hCG CollectedBefore the Onset of Preeclampsia

Ninety-six-well microtitre plates (Maxisorp, Nunc) were coated with 50μI rabbit anti-human hCG antibody (5 μ9/ιηI in PBS, Dako A0231) at 4° C.overnight. The wells were washed three times with PBS, pH 7.2 containing0.05% Tween 20, blocked for 1 hour with washing buffer containing 1% BSAand washed again three times.

50 μI of human serum obtained from blood collected at 12-14 weeks ofpregnancy who later either went on have normal pregnancy (n=8) or werediagnosed with preeclampsia (n=8) were added and incubated for 1.5 hoursat room temperature and washed three times. 50 μI of sLe^(x)(Calbiochem. KM93) or sLe^(a) (Calbiochem, KM 231) or Le^(y) orThomsen-Friedenreich (TF) antigen (Glycotope) recognizing antibodieswere added at concentration of 5 μg/ml in PBS.

The wells were incubated for 1.5 hours at room temperature and washedthree times. 50 μI of HRP-conjugated rabbit anti-mouse antibody (Dako,PO260) was added to each well, incubated for 1.5 hours, washed threetimes, developed with DMB and color development was followed bymeasuring the absorbance at 492 nm/630 nm. The mean absorbance obtainedwith multiple normal pregnancy hCG were considered as 100%.

As seen in the FIG. 3, sialyl Lewis antigen (sLe^(a) and sLe^(x))expression is significantly elevated on preeclampsia serum (PES)-hCGcollected at 12-14 weeks of pregnancy before the clinical diagnosis ofdisease as compared to normal pregnancy serum (NPS)-hCG. The increase insialyl Lewis antigen expression in PES-hCG was independent of the serumlevels of β-hCG. 11.2% upregulation in SLe^(a) and 22.4% upregulation inSLe^(x) expression in the 10th-12th week of gestation in preeclamptichCG compared to normal pregnancy hCG. The expression of the TF antigenis not significantly changed in preeclamptic hCG compared to normalcontrol hCG.

Example 4 Rescue of Preeclampsia-Like Features (IUGR, Hypertension andProteinuria) by Sialyl Lewis Antigen-Free hCG (Functional hCG) in MouseModel

Pregnant IL-10⁷″ mice were injected (gestational day 10, i.p) witheither normal pregnancy serum (NPS) or PE serum (PES) with or withoutsialyl Lewis antigen-free hCG (urine or recombinant).

On gestational day 17, blood pressure and fetal weight were recordedUrinary albumin and creatinine was measured in 24-hour urine samplesusing commercial ELISA kits. Proteinuria is expressed as a ratio ofalbumin and creatinine.

As seen in FIG. 4, functional hCG treatment reverses PES-inducedintrauterine growth restriction (IUGR) (A) as reflected by fetal size(upper panel) and fetal weights (lower panel), hypertension (B), andproteinuria (C) in pregnant IL-10⁷″ mice. * and ^(a)P<0.05 significanceas compared to NPS and PES groups respectively by student's T test

Example 5 Rescue of Preeclampsia-Like Features (Kidney Pathology,Elevated Soluble Fit-1 and Soluble Endoqiin) by Sialyl LewisAntigen-Free hCG (Functional hCG) in Mouse Model

Pregnant IL-10⁷″ mice were injected (gestational day 10, i.p) witheither NPS or PES with or without sialyl Lewis antigen-free hCG (urineor recombinant).

On gestational day 17, blood was collected by cardiac puncture and serumseparated. Serum levels of mouse sFlt-1 & sEng were measured usingcommercial ELISA kits (R&D Systems).

As seen in FIG. 5, functional hCG treatment reverses PES-induced renalpathology as shown by H&E staining of glomerulus (A), and excessproduction of sFlt-1 (B) and sEng (C) in pregnant IL-10⁷″ mice. * and^(a)P<0.05 significance as compared to NPS and PES groups respectivelyby student's T test

Example 6 Treatment of Pregnant Human with Sialyl Lewis AntigenElevation Before 20 Weeks of Pregnancy

A 26 year old female presents at 10 weeks of pregnancy. Her serum istested by the method of Example 2. The test detects sialyl Lewis antigensLe^(Y) levels above 25% average control normal.

These results are consistent with and predictive of consistent withincipient PE. She is then dosed with recombinant 100 IU hCG, i.v. Thepregnancy comes to term without either insufficient trophoblast invasionor marked maternal spiral artery remodeling and inflammation.

Example 7 Treatment of Pregnant Human with Sialyl Lewis AntigenElevation Before 20 Weeks of Pregnancy

A 26 year old female presents at 10 weeks of pregnancy. Her serum istested by the method of Example 2. The test detects sialyl Lewis antigensLe^(Y) level of 18% above average control normal, and sLe^(x) level of18% above average control normal, and sLe^(a) level of 15% above averagecontrol normal, with a combined percentage of over 50% above averagecontrol. These results are consistent with and predictive of incipientPE. She is then managed for preeclampsia. The pregnancy comes to termwithout either insufficient trophoblast invasion or marked maternalspiral artery remodeling and inflammation.

The invention claimed is:
 1. A method of treating incipient preeclampsiain a human-female patient-subject by (a) determining the level of sialylLewis antigen A in said patient-subject at about 10 weeks of pregnancyor earner; (b) determining that said level of sialyl Lewis antigen A ofstep (a) is at or greater than about 11.2% above a normal control level;and (c) administering to said patient-subject a therapeuticallyeffective amount of human chorionic gonadotropin (hCG): thereby treatingthe human female patient-subject for incipient preeclampsia.
 2. Themethod of claim 1, further comprising: prior to administering the hCG ofstep (c), managing said patient-subject for preeclampsia.
 3. The methodof claim 1, wherein the hCG is recombinant hCG.
 4. The method of claim1, wherein the therapeutically effective amount of hCG is administeredintravenously.
 5. The method of claim 1, wherein the therapeuticallyeffective amount of hCG is from between about 50 I.U. and about 500 I.U.6. The method of claim 5, wherein the therapeutically effective amountof hCG is from between about 100 I.U. and about 200 I.U.